Anti-CD3 antibody-aminodextran conjugates for induction of T-cell activation and proliferation

ABSTRACT

The invention describes the use of novel aminodextran compounds containing about 5-20% by weight amine groups to bind a plurality of monoclonal antibodies. The resulting antibody-aminodextran compounds may be used to induce the activation and proliferation of selected mammalian cells. Specific examples are given using an anti-CD3 monoclonal antibody conjugated two aminodextrans containing about 5% and 16%, respectively, by weight amine groups as an agent for inducing the activation and proliferation of T cells.

This is a continuation of application Ser. No. 08/075,647 filed on Jun.11, 1993 now abandoned.

RELATED INVENTIONS

This application is related to U.S. Pat. No. 5,248,772 entitledFORMATION OF COLLOIDAL METAL DISPERSIONS USING AMINODEXTRANS ASREDUCTANTS AND PROTECTIVE AGENTS, issued Sep. 28, 1993; and co-pendingapplications entitled POLYMERIC PARTICLES HAVING A BIODEGRADABLE GELATIN0R AMINODEXTRAN COATING AND PROCESS FOR MAKING SAME, Ser. No.07/961,157, filed Oct. 15, 1992; and BIODEGRADABLE GELATIN-AMINODEXTRANPARTICLE COATINGS AND PROCESS FOR MAKING SAME, Ser. No. 07/968,158,filed Oct. 29, 1992, which is a Continuation-In-Part of U.S. Pat. No.5,169,754, issued Dec. 8, 1992, entitled BIODEGRADABLE PARTICLE COATINGHAVING A PROTEIN COVALENTLY IMMOBILIZED BY MEANS OF A CROSSLINKING AGENTAND PROCESS FOR MAKING SAME. This application and the relatedapplications and patent are wholly owned by a single common assignee,Coulter Corporation, Miami, Fla. The teachings of these patents andcopending applications are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to the use of novel antibody-aminodextranconjugates for use in inducing the activation and proliferation ofleukocytes. In particular, the invention relates to the use of ananti-CD3 monoclonal antibody covalently coupled to an aminodextran. Thecovalently coupled antibody-aminodextran conjugates have application inthe analysis of immune cell functions in patients with various medicalconditions, such as AIDS and other immunodeficiencies, infectiousdiseases, cancers, autoimmunity, and atopic diseases. In addition, theleukocytes from recipients of transplanted cells and/or tissues can befunctionally evaluated utilizing this technology.

BACKGROUND OF THE INVENTION

Immunosuppression, whether induced by drugs or disease, can lead toalterations in T cell and/or accessory cell function. For example, ithas been demonstrated that AIDS patients manifest defective responses tomitogens, autoantigens, alloantigens, and soluble antigens. This alteredimmunoreactivity is attributable to defects in both responding (T) andstimulating (monocyte and dendritic) cell populations. Decreased CD4expression on the monocytes obtained from AIDS patients has beendemonstrated, yet no decrease in monocyte count has been observed.Immunosuppressive drugs can also alter antigen presenting cell function.As a result of these observations, it has been determined that a methodfor specifically stimulating T cells which did not rely on the presenceof monocytes or dendritic cells would be desirable.

While there are numerous methods of activating T cells, the optimalmethod appears to require the multivalent interaction of antibodies, orother receptor binding species such as lectins, with the T cell antigenreceptor/CD3 complex [hereafter TCR/CD3] on the surface of T cells. [A.Altman et al., Crit. Revs. in Immunol. 10:347-391 (1990)]. CD3 specificmonoclonal antibodies can induce highly purified, resting T cells toproliferate, provided, however, that there is present a mechanism forcrosslinking the antibody-bound TCR/CD3 complexes. Numerous authors haveshown that the crosslinking requirement can be met by binding theantibody to a substrate, for example, Sepharose® beads [S. Meuer et al.,J. Exp. Med. 158:988-999 (1983) and D. A. Hafler et al., J. Immunol.142:2590-2596 (1989)], polystyrene beads [S. Panzer et al., Scand. J.Immunol. 32:359-371 (1990)], or tissue culture dishes. InternationalPatent Publication WO 90/04633 describes solid-state supportedmonoclonal antibodies for induction of T cell activation and the growthof T cells. Accessory cells, for example, monocytes, can also fulfillthe need for crosslinking by means of Fc receptor mediated binding ofthe T cell bound anti-CD3 monoclonal antibodies to the monocyte cellsurface.

The parameters used to assess cellular activation were reviewed by A.Altman et al., Crit. Revs. in Immunol. 10: 347-391 (1990). Cellactivation has been measured by changes in nucleic acid synthesis,protein or glycoprotein synthesis, cellular size and morphology,membrane integrity, expression of cellular constituents, cell function,cell growth, cell differentiation and the release of cellularcomponents. These cellular changes have been detected by numerousdifferent methods, many of which are described in the patents andpublications cited herein. Historically, the diagnosis of immunedeficient conditions has been done using laboratory tests in whichvarious stimuli are applied to T cells in order to determine if they canbe activated in vitro. Deficient T cell reactivity has been tested bystimulation with mitogens, alloantigens and soluble antigens [R. Hong inManual of Clinical Immunology, 2nd Ed., N. R. Rose and H. Friedman, eds.(American Society for Microbiology 1980), Chapter 111, pages 833-849].However, these methods are not specific for all T cells. Mitogensactivate both T and B cells. Alloantigens activate only those selected Tcells which have the appropriate receptor type. The response to solubleantigens, for example, tetanus toxoid, can be effected by a patient'simmunization history. The anti-CD3 aminodextran conjugates of theclaimed invention specifically activate all CD3 positive cells and thusavoid these problems. The CD3 antigen is found on virtually all matureperipheral T lymphocytes. This antigen is a component of the T cellreceptor complex and is non-covalently linked to a polymorphic,clonotypic structure termed Ti. Antibodies to the CD3 surface structureserve as probes for constant regions of the T cell receptor which isexclusively expressed on immunocompetent T lymphocytes. Consequently,quantifying immunocompetent T cells using this antibody is rapid andefficient.

In addition to the forementioned copending applications, particlescoated with dextran or dextran derivatives have been described by R. J.Mrsny et al., Eur. J. Cell. Biol. 45:200-208 (1987)(ouabain-aminodextran-gold particles); J. W. M. Bulte et al. Magn.Reson. Med. 25:148-157 (1992) (biotinylated dextran-magnetiteparticles). The use of antibody-dextran type substances as carriers hasbeen described by U. Manabe et al., J. Lab Clin. Med. 104:445-454 (1984)(antibody-polyaldehyde dextran-methotrexate); A. R. Oseroff et al.,Proc. Natl. Acad. Sci. USA 83:8744-8748 (1986)(antibody-aminodextran-chlorin); and S. Rakestraw et al., Proc. Natl.Acad. Sci. USA 87:4217-4221 (1990) (antibody-dextran hydrazide-Sn(IV)chlorin). Other conjugated and crosslinked species have been describedby S. S. Wang in "Chemistry of Protein Conjugation and Crosslinking"(CRC Press, Boca Raton, Fla. 1991) and H. Maeda et al., BioconjugateChem. 3:351-362 (1992). The standard procedure for the introduction ofamine groups into dextran has been to first cleave the sugar rings toform polyaldehyde-dextran. The second step is to react the cleaved ringswith a diamine such as ethylenediamine or 1,3-diaminopropane to form aSchiff's base complex. The Schiff's base is then stabilized by reductionwith sodium borohydride. The "aminodextran" compounds as described inthe above cited art were ill-described, typically lacking eitherelemental analyses or even average molecular weight determinations.Furthermore, the periodate oxidation method of preparing aminodextransas described in these publications resulted in a low percentage of aminogroups per molecule. The percentage was less than 4-5 percent. Higherdegrees of amine substitution were not possible under the usualconditions of the prior art because high diamine concentrations causedextensive aminolysis of the glucosidic linkages between the sugar ringsin dextran which resulted in very low molecular weight fragments. As aresult, the yields of polymeric aminodextran derivatives were low anddecreased drastically as higher and higher degrees of amine substitutionwere pursued.

An alternative method of producing aminodextrans is bycarboxymethylation of sugar residue hydroxyl groups in chloroaceticacid, followed by carbodiimide coupling of a diamine such asethylenediamine. M. Brunswick et al., J. Immunol 140:3364-3372 (1988)and P. K. A. Mongini et al., J. Immunol. 148:3892-3902 (1992) used thismethod to produce an aminodextran having about one amine group persixty-seven glucose residues (1/67). These authors then used theaminodextrans to prepare anti-Ig antibody-aminodextran conjugates foruse in inducing B cell activation and proliferation.

The claimed invention teaches the use of anti-CD3 monoclonal antibodiesconjugated to aminodextrans as a method of providing for the specificstimulation of T cells. Analysis of T cell function is critical to thediagnosis of immunodeficiency. For example, the CD3-aminodextranconjugates described herein provide a uniquely specific method foractivating the T cells used for T cell analysis in AIDS patients. M.Clerci et al., J. Clin. Invest. 84:1892-1899 (1988) found that an invitro T helper cell (T_(H)) assay "can detect multiple stages of immunedysregulation early in the course of HIV infection". Tetanus toxoid andalloantigens, which have selectivity problems as stated above, were usedas stimuli for T cells. S. C. Muluk et al., Transplantation Proceedings23:1274-1276 (1991) have shown that T cell monitoring can be useful fordetermining the efficacy of immunosuppressive agents in transplantpatients.

The claimed invention teaches the use of aminodextrans, particularlyaminodextrans having a high degree or percentage of amine substitution,as a means of crosslinking antibodies and of using the resultingantibody-aminodextran conjugate to induce activation and proliferationof T cells. Aminodextrans have been used in the copending applicationscited herein to coat polystyrene microspheres and magnetic andnon-magnetic particles such as ferrite and metallic gold particles. Theaminodextran coated particles are then used to covalently link variousmonoclonal antibodies. Both the degree of amine substitution and thedegree of polymerization of the dextran can be varied to determine theoptimal form of the resulting coated particle to which an antibody canbe conjugated. Non-specific interactions between antibody-aminodextrancoated particles and cells are minimized by blocking amine groups withexcess crosslinking agent which, in turn, is also blocked.

SUMMARY OF THE INVENTION

The invention relates to the use of aminodextrans having 5-20% by weightamine groups as crosslinking agents for monoclonal antibodies to produceantibody-aminodextran conjugates that are useful in the induction andactivation of mammalian cells and especially human cells. In particular,the conjugates find utility for inducing the activation andproliferation of human T and B cells. A preferred embodiment of theinvention is the preparation and use of anti-CD3 monoclonal antibodiesconjugated to aminodextrans to induce T cell activation andproliferation. The invention further describes the use of novelaminodextrans having a high degree of amine substitution (greater than10%) in the formation of such conjugates and compares the results withconjugates formed using aminodextrans generally known in the art whichtypically have about 4-5% amine substitution. Comparative resultsindicate that the use of aminodextrans with high amine content ispreferred.

The invention also relates to a method of analyzing mammalian T cells,particularly human cells. A sample containing or thought to contain Tcells is reacted with an aminodextran/anti-T-cell monoclonal antibodyprepared as described herein. The resulting aminodextran-antibody-cellcomplex, after any appropriate incubation time, may be analyzed toevaluate T cell functions or changes in T cell function. Tests which maybe used in such analysis comprise changes in nucleic acid (DNA or RNA)synthesis, protein or glycoprotein synthesis, cellular size andmorphology, membrane integrity, expression of cellular constituents andthe release of cellular components into the medium containing the cellsundergoing analysis. Typical diseases or disorders which are amenable tothis type of analysis are AIDS, other non-AIDS immunodeficiencydiseases, infectious diseases, cancer, autoimmunity disorders and atopicdisorders. The method also may be used to test T cells from patients whoare the recipients of tissue, organ or cell transplants. In those casesinvolving transplants, prior to the T cell analysis, non-T leukocytecells and their immature precursors may be stimulated to facilitateadditional testing related to conditions arising from the transplant.The non-T cells include B cells, macrophages/monocytes, dendritic cells,neutrophils, eosinophils, basophils, cytotoxic effector cells,hematopoietic stem cells and the immature precursor cells of each ofthese cells. Methods stimulating such non-T cells and their precursorcells are described in the publications cited in this application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the distribution of cells in the forward versus sidescatter histogram of a sample containing unactivated, control T cells.

FIG. 2 illustrates the distribution of cells in the forward versus sidescatter histogram of a sample containing T cells activated by the use ofan anti-CD3/1X-Amdex conjugate.

FIG. 3 illustrates the distribution of cells in the forward versus sidescatter histogram of a sample containing T cells activated by the use ofan anti-CD3/5X-Amdex conjugate.

FIG. 4 graphically compares the effect of selected anti-CD3/Amdexconjugates on the formation of activated blast cells.

FIG. 5 illustrates the DNA content of control cultured T cells whichwere not stimulated with anti-CD3/Amdex conjugates.

FIG. 6 illustrates the DNA content of T cells activated withanti-CD3/1X-Amdex conjugates.

FIG. 7 illustrates the DNA content of T cells activated withanti-CD3/5X-Amdex conjugates.

FIG. 8 graphically illustrates the effect of selected anti-CD3/Amdexconjugates on the percentage of CD25, CD71, and PCNA positive cells.

DETAILED DESCRIPTION OF THE INVENTION

Crosslinked antibodies show enhanced ability to induce resting T cellsin the G₀ /G₁ phase to progress into the readily observable S,G₂ /Mphases of the cell cycle, including DNA synthesis. Soluble polymers suchas aminodextran, which have been used to coat a variety of colloidalparticles, should be suitable crosslinking agents for antibodies used toinitiate and sustain T cell proliferation. While aminodextrans have beenused in conjugates keyed to the stimulation of B cells, they have notbeen used with T cells. We describe herein improved methods forpreparing aminodextrans, particularly those with greater than 10% aminesubstitution, and their use in the formation of antibody-aminodextranconjugates which are subsequently used for stimulating the activationand proliferation of mammalian cells and especially for stimulating Tcell activation and proliferation. Both the degree of amine substitutionand the degree of dextran polymerization can be varied to find theoptimal form of the aminodextran to be used as an antibody carrier andcrosslinking agent.

The examples provided herein are for illustrating the invention and arenot to be construed as limiting the invention. For example, while theexamples herein describe the induction of human T cell activation andproliferation, the proper choice of antibodies may extend the utility toother cells, such as a B cell, and other mammalian species, such as acat, dog or horse.

Two forms of aminodextran were used to form the conjugates describedherein. The first, which is described below as 1X-aminodextran(abbreviated 1X-Amdex), has a degree of substitution equal to about 1/32(two 1,3-diaminopropane groups per sugar residue) and an averagemolecular weight of about 1,000,000 daltons. The second, described belowas 5X-aminodextran (abbreviated 5X-Amdex), has a degree of substitutionequal to about 1/7 and an average molecular weight of about 350,000daltons.

The anti-CD3 monoclonal antibody used herein was obtained from CoulterCorporation, Miami, Fla. and was activated for conjugation to theaminodextran by iminothiolane using standard procedures. The source ofthe anti-CD3 monoclonal antibody or other T cell activating monoclonalantibodies is not critical to the invention and other sources of suchantibody may be used in place of that described herein. Theaminodextrans were activated with the heterobifunctional reagentsulfo-SMCC [sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate] prior to conjugation with the activatedantibody. The invention is not limited to the use of iminothiolane andsulfo-SMCC as activating agents. Those skilled in the art will recognizethat other reagents, such as those described in U.S. Pat. No. 5,169,754and the other related applications, may be used in place of theiminothiolane and sulfo-SMCC used herein.

The Methods A-C described herein and in copending application Ser. Nos.07/968,158 and 07/827,347 may be used to prepare aminodextrans having anamine content in the range of greater-than-zero to about twenty percent.Method C is preferred for preparing aminodextrans having more than 5%amine groups; particularly, for aminodextrans having more than 10% aminegroups. The use of the hollow fiber cartridge described herein places alower molecular weight limit of 5,000 daltons on the aminodextransprepared using such cartridge. This lower limit may be raised or loweredby changing the choice of cartridge used in the process. The maximummolecular weight of the aminodextran products is limited to that of thestarting dextran materials. Aminodextrans prepared by oxidative cleavagemethods known in the art have a maximum of 4-5% amine group. The methodsdescribed herein allow for the preparation of aminodextrans having 300%to 400% more amine groups than the aminodextrans described in the art.

Antibodies other than the Coulter T3 antibody described herein have alsobeen used according to the invention. These include an anti-CD2 antibody(Coulter T11, IgG1), and an antibody against an 85,000 dalton speciesexpressed on the gamma chain of the T cell receptor (Coulter TiGamma,IgG1) among others. An anti-CD3 monoclonal antibody such as T3 ispreferred.

When the invention is used in conjunction with methods of stimulatingnon-T cells and their precursor cells the methods used are those foundin the technical literature and known to those skilled in the art. Forexample, if one wishes to analyze and/or evaluate B cells as well as Tcells, one may use anti-IgD and anti-IgM monoclonal antibodies asdescribed by M. Brunswick et al. in J. Immunology 140: 3364-3372 (1988).In such a case, the dextran or Ficoll used by Brunswick et al. may bereplaced by the aminodextrans disclosed herein. If one wished tostimulate and activate macrophages, one may use a poly(styrene-comaleicn-butyl ester)-conjugated neocarzinostatin as taught by H. Maeda et al.in Bioconjugate Chemistry 3: 351-362 (1992).

I. Preparation of Aminodextrans.

Method A. Small scale preparation of aminodextran.

Aminodextran was prepared by partial cleavage and oxidation of theglucopyranose rings in dextran to give aldehyde functional groups,coupling of the aldehyde groups with 1,3- diaminopropane to form Schiffbase linkages and reduction of the Schiff's base linkages to form stablecarbon-nitrogen bonds. In a typical procedure, 20 g of dextran weredissolved in 150 ml of 50 mM potassium acetate buffer, pH 6.5. Asolution of 2.14 g of sodium periodate in 25 ml of distilled water wasadded dropwise to the dextran over about 10 minutes using vigorousmagnetic mixing. The resulting solution was stirred at room temperature,15°-27° C., for about 1.5 hours and then dialyzed against distilledwater. 20 ml of 1,3-diaminopropane were mixed with 20 ml of distilledwater, cooled in an ice bath, vigorously stirred and pH adjusted fromabout 11.5 to about 8.7 over about 15 minutes by the addition of glacialacetic acid. Typically, 15-20 ml of glacial acetic acid were used. Thedialyzed dextran solution was added dropwise over about 15-20 minutes tothe chilled diamine solution. After the addition was completed, theresulting solution was stirred at room temperature for about 2.25 hours.A reducing solution of 0.8 g sodium borohydride in 10 ml of 0.1 mMsodium hydroxide was added to the dextran reaction mixture at roomtemperature over about 15 minutes. The reaction mixture was stirredduring the borohydride addition to expel most of the effervescence. Thecrude aminodextran solution was exhaustively dialyzed against distilledwater until the conductivity of the effluent was 3-4 μmho/cm. Thedialyzed solution was then filtered through a 0.2 μm filter andfreeze-dried over 24 hours in a model TDS-00030-A, Dura-Dry®microprocessor controlled freeze-dryer (FTS Systems, Inc.) to produce4.25 g of flaky, pale yellow crystals in 21% yield.

Method B. Large scale preparation of aminodextran.

The procedure of Method A was modified for the large scale preparationof aminodextran and for increasing the number of amine groups introducedinto dextran. Hollow fiber membrane filtration replaces dialysis and asmaller diamine-periodate molar ratio was used to avoid further cleavageof the sugar polymer into lower molecular weight fragments. Thesechanges also serve to shorten the contact time between excess diamineand substituted dextran during the removal of excess low molecularweight reagents. Without these changes, the aminolysis of the glucosidiclinkages in high molecular weight dextran (e.g. dextran T-2M) was veryextensive and drastically reduced the yield of aminodextran above acut-off molecular weight of 5,000 daltons. A hollow fiber cartridge(polysulfone, 3 ft² membrane surface area, 1 mm diameter fibers and5,000 MW cut-off model UFP-5-E-6, A/G Technology Corp.) was mountedvertically with an input power pump (two pump heads, maximum flow rateof about 4.56 liters/minute with No. 18 Norprene® food grade tubing)delivering 15-20 psi which corresponds to 5-10 psi in the retentateline. The filtrate was collected at 50-100 ml/min. Washing was doneusing 20-30 liters of distilled water over about 6-8 hours. The specificconductance was reduced to about 3-4 μmho-cm⁻¹ and the pH was 6.0-6.5.The feed volume was maintained at 2 liters during desalting and thenconcentrated to 800 ml in the first washing of oxidized dextran and to400 ml in the second washing of The feed volume was maintained at 2liters during desalting

In a standard scaled-up preparation, 80 g of dextran were transferred to1 quart [liter] glass blender bowl containing 600 ml distilled water.The solid was blended for about 2-5 minutes at medium speed to dissolveall the dextran. 8.56 g of sodium periodate were dissolved in 100 ml ofdistilled water and the resulting solution was added dropwise to thedextran solution over about 10 minutes using vigorous magnetic stirring.After the addition was completed, the resulting mixture was stirred atroom temperature for an additional 3 hours. The resulting viscousreaction mixture was then diluted to 2 liters with distilled water anddesalted using a hollow fiber cartridge. The initial specificconductance was 1.5 mmho-cm⁻¹ or higher and the initial pH was 4.0.About 18-22 liters of distilled water were used to obtain a solutionhaving a final pH of 6.0-6.5. The final volume of washed, oxidizeddextran solution was 800 ml.

To the washed, oxidized dextran solution, 80 ml of colorless, liquid1,3-diaminopropane were slowly added over about 10 minutes at roomtemperature. The resulting mixture was then stirred at room temperaturefor an additional 3 hours. After the stirring was finished, 3.2 g ofsodium borohydride dissolved in 40 ml of 1 mM aqueous sodium hydroxidewere added to the room temperature aminodextran reaction mixture overabout 5 minutes with magnetic stirring. After the completion of thesodium borohydride addition, the resulting mixture was stirred for anadditional 1 hour and then desalted using a hollow fiber cartridge. Theinitial specific conductance was 5.0 mmho-cm⁻¹ or higher and the initialpH was about 12.0. About 20-25 liters of distilled water were needed toreduce the specific conductance to about 3-4 μmho-cm⁻¹ and the pH to6.0-6.5. The final volume of aminodextran solution was 400 ml. Thissolution was passed through a 0.2 μm sterile cellulose acetate filterunit and then freeze-dried over 48 hours to obtain 48 grams of flaky,pale yellow crystals, a 52% yield.

Elemental analyses (C,H,N) were obtained for two samples of aminodextranprepared from dextran T-2M by the methods described above. The analysesare:

Sample 1. 20 g dextran scale, Method A (desalting by dialysis). Obsd.:C, 43.04; H, 6.60, N, 1.09; O (by difference), 49.27. Calculated for C₄₆H₇₉ NO₃₇.3H₂ O: C, 42.76; H, 6.63; N, 1.08; O, 49.53.

Sample 2. 80 g dextran scale, Method B (desalting by membranefiltration). Obsd.: C, 42.53: H, 6.52N, 1.01; O (by difference), 49.94Calculated for C₄₉ H₈₄ NO₄₀.3H₂ O: C, 42.61; H, 6.57; N, 1.01; O, 49.81

The analyses for aminodextran in the two preparations were very similar,thus indicating that the same product was obtained whether desalting wasdone by dialysis or by membrane filtration and whether acetate bufferwas used or not used. The yield of aminodextran, however, was raised by31% in Method B over that in Method A. The empirical formula obtainedfor Sample 1, C₄₆ H₈₄ NO₄₀, is very similar to the formula C₄₆ H₇₉NO₃₇.3H₂ O based on 29 units of glucose (C₆ H₁₀ O₅), 1 unit of fullydiamine-substituted sugar ring (C₁₂ H₂₈ N₄ O₃ two mol diamine per molsugar unit,) and twelve units of water. Therefore, the degree of diaminesubstitution of sugar residues in dextran was 1/30 in Sample 1 incontrast to a theoretical value of 1/12 based on 100% periodate cleavageand diamine substitution according to the balanced oxidation-reductionequation. The empirical formula obtained for Sample 2, C₄₉ H₉₀ NO₄₃, isvery similar to the formula C₄₉ H₈₄ NO₄₀.3H₂ O based on 31 units ofglucose, 1 unit of fully diamine substituted sugar ring and twelve unitsof water. The degree of substitution in dextran by diamine was 1/32 forSample 2.

Similar results were obtained using aminodextrans having averagemolecular weights of 10,000, 40,000 and 2,000,000 daltons (T-10, T-40and T-2M) with 1X (1X=3.3% substitution of sugar residues), 2X (6.6%) 3X(9.9%) and 5X (16.5%) molar amounts of amino groups. All theaminodextrans were initially prepared according to methods A and B using2 and 3 times the amount of sodium periodate used in the 1X oxidation ofdextran. The amount of 1,3-diaminopropane used for Schiff base formationwas kept constant.

Modifications have been made to the Methods A and B of preparingaminodextrans which were originally disclosed in Ser. No. 07/827,347.These modifications, disclosed in Ser. No. 07/968,158, involve theoxidation and cleavage of the dextran glucose rings with periodateanion, diamine addition and sodium borohydride reduction of the Schiff'sbase. The modifications have resulted in increased yield of theaminodextrans, particularly the 5X-aminodextran which was produced inless than 5% yield by the old procedures. Generally, the firstmodification was to use only a ten percent (10%) excess of diamine overthe stoichiometric 2:1 diamine:periodate molar ratio previouslydisclosed. Second, the diamine addition reaction was conducted at atemperature in the range of about 5°-10° C. Third, the diamine additionreaction was spectroscopically monitored in the near ultraviolet (UV)region for Schiff base formation. Schiff's base formation was deemedcompleted when successive spectral analyses indicated a plateau wasreached. The reaction was then quenched by sodium borohydride additionwhich reduces the Schiff's base linkages to carbon-nitrogen single bondsand reduces any unreacted aldehyde groups to alcohol groups. Thesemodifications reduced aminolysis of the polymeric sugar groups intolower weight fragments and thus gave higher yield of product afterpurification and concentration by hollow fiber membrane filtration. Thehollow fiber filtration was done using polysulfone cartridge of 3 ft.²membrane surface area, 1 mm diameter fibers having a 5,000 molecularweight cut off. The cartridge was mounted vertically in an input powerpump having two pump heads delivering 15-20 psi with a maximum flow rateof 4.56 liter/minute when using No. 18 Norprene® food grade tubing. Withthis configuration, the pressure in the retenate line was about 5-10psi. The filtrate was collected at 50-100 ml/min. Washing was done using20-30 liters of distilled water over about 6-8 hours. The followingmethod for preparing 5X-aminodextran is given to illustrate the modifiedprocedure which is applicable to the preparation of all aminodextrans.

Method C. Preparation of 5X-Aminodextran.

T-2M dextran (50g, 0.308 mol, obtained from Sigma, St. Louis, Mo., orPharmacia, Piscatawny, N.J.) was added to a 1-quart or 1-liter glassblender bowl containing 300 ml of distilled water. The mixture wasblended at maximum speed until all the dextran dissolved, typicallyabout 3-5 minutes. A solution of 26.75 g (0.125 mol) of NaIO₄ in 300 mldistilled water was added to the dextran solution over about a 10 minuteperiod using vigorous magnetic stirring. After the periodate additionwas completed, the reaction mixture was stirred at room temperature forabout an additional three hours. After the three hours, the 600 mlreaction volume had an initial specific conductivity of 9.7 mmho-cm⁻¹and an initial pH of 2.5. The reaction mixture was diluted to two literswith distilled water and desalted using the hollow fiber cartridge.Washing was done using 15-18 liter of distilled water to obtain 600 mlof washed, oxidized dextran solution having a specific conductance of 10μmho-cm⁻¹ and pH of 6.7.

The solution of oxidized dextran was cooled to about 8° C. using an icebath and 23.2 ml (0.275 mol) of 1,3-diaminopropane was added over about10 minutes to the oxidized dextran solution. The resulting reactionmixture was stirred and maintained at the ice bath temperature. Theformation of the yellow Schiff's base was monitored ever 10-15 minutesby measuring the 335 nm near-UV absorbance of an extracted sample. In atypical experiment, the measurements at 335 nm using a 1 mm path lengthcell were:

                  TABLE 1                                                         ______________________________________                                        minutes      absorbance values                                                ______________________________________                                        0            0.100                                                            5            2.063                                                            15           2.975                                                            30           3.692                                                            45           3.901                                                            60           4.103                                                            75           3.784                                                            ______________________________________                                    

After the absorbance had reached a plateau, 19.3 g (0.500 mol) of sodiumborohydride in 19.3 ml of 1 mM aqueous potassium hydroxide were added tothe reaction mixture over about 10 minutes at ambient room temperatureusing magnetic stirring. After the sodium borohydride addition wascompleted, the reaction mixture was stirred at ambient room temperaturefor about an additional two hours. After the stirring was completed,spectroscopic measurement at 335 nm using a 1 cm path length cell gavean absorbance value of 0.067 units which indicates that the Schiff'sbase compound had essentially disappeared. The reaction mixture, about1000 ml volume, was then desalted using the hollow fiber cartridge. Theinitial specific conductance was 43 mmho-cm⁻¹ and the initial pH was11.0. About 18-20 liters of distilled water were used as wash liquid toproduce about 300 ml of 5X-aminodextran solution having a specificconductance of about 4-6 μmho-cm⁻¹ and a pH of 6.5-7.0. The5X-aminodextran solution was filtered through a 0.2 μm cellulose nitratefilter and freeze-dried over 48 hours in a model TDS-00030-A, Dura-Dry®microprocessor-controlled freeze-dryer (FTS Systems, Inc.) to produce 24g (48% yield) of flaky, pale yellow crystals. Elemental analysis:C=45.83%, H=7.00%, N=4.49%, O (by difference)=42.68%. Calculatedanalysis for C₁₂ H₂₂ O₈.25 N: C=46.15%, H=7.10%, N=4.48%, O=42.26%.

The empirical formula based on actual analysis is C₁₂ H₂₂ O₈.3 N, whichis very similar to the formula C₁₂ H₂₂ O₈.25 N based on 6 units ofglucose per one unit of fully diamine-substituted sugar ring (C₁₂ H₂₈ N₄O₃). Therefore, the degree of diamine substitution in dextran was 1/7 incontrast to a theoretical value of 1/2.5 based on 100% periodatecleavage and diamine substitution.

Repeat experiments using an initial charge of 100 g and 300 g dextranproduced a 5X-Amdex product having a similar degree of substitution. Thepreparation of 1X-Amdex was also scaled up to the 100 g and 300 g levelsusing Method C. At the 300 g scale the hollow fiber cartridge waschanged to a polysulfone membrane of 8.5 ft.³ surface area, 1 mmdiameter fibers having a 5,000 nominal molecular weight cut-off (modelUFP-5-E-35, A/G Technology Corp.). The cartridge was used forultrafiltration of both the oxidized dextran and the final aminodextranproduct to remove salts and low molecular weight reagents orby-products. At the 300 g scale, the yields of the 5X-Amdex and 1X-Amdexproducts were 135 g (45%) and 162.1 g (57%), respectively. The elementalanalysis of the products were: 1X-Amdex, 300 g dextran scale. Obsd.: C,43.58; H, 6.50; N, 0.82; O (by difference), 49.10 Calculated for C₆₂H₁₀₅ NO₅₀.2H₂ O: C, 43.79; H, 6.46; N, 0.82; O, 48.92 5X-Amdex, 300 gdextran scale. Obsd.: C, 45.67; H, 6.90; N, 4.04 O (by difference),43.39 Calculated for C₁₃ H₂₄ O₉ N: C, 46.15; H, 7.15; N, 4.14; O, 42.56The empirical formula obtained for 5X-Amdex, C₁₉ H₂₄ NO₉, is verysimilar to the formula C₁₃.2 H₂₄ NO₉.5 based on 68 units of glucose andone unit of fully diamine-substituted sugar ring. The degree ofsubstitution of sugar residues in dextran was, therefore, about 1/8. Theempirical formula C₆₂ H₁₁₀ NO₅₂ obtained for the 1X-Amdex is similar tothe formula C₆₂ H₁₀₅ NO₅₀.2H₂ O based on 39.3 units of glucose, one unitof fully diamine-substituted sugar ring and two units of water. Thedegree of substitution of sugar residues in the dextran was, therefore,about 1/40.

II. Preparation of Anti-CD3 Antibody-Aminodextran Conjugate

1. Activation of aminodextran with sulfo-SMCC.

25 mg of 1X-Amdex and 5X-Amdex were dissolved in 6.667 ml portions of 1XPBS in separate, sealable 15 ml tubes to give solutions having aconcentration of 3.75 mg/ml. 1X PBS is made by dilution of a 20X PBSsolution comprising 53.89 g K₂ HPO₄, 12.8 g KH₂ PO₄ and 340 g NaCl in 2L distilled water as described in copending application Ser. No.07/961,057. The 1X-Amdex was activated by the addition of 13.5 μL of 10mg/ml sulfo-SMCC solution per milliliter 1X-Amdex solution (a total of0.090 ml sulfo-SMCC solution). The 5X-Amdex was activated using fivetimes the amount of sulfo-SMCC solution (0.450 ml). The sulfo-SMCC waspipetted into the respective tubes, vortexed to mix well and then rollermixed for about two hours. After the mixing was completed, each reactionmixture was chromatographed on a separate 100 ml G-50 Sephadex® column(2.5 cm×20 cm) equilibrated with 1X PBS. The samples were eluted using1X PBS and collected in about 4 ml fractions. Fractions of the firstband absorbing at 280 nm contain the high molecular weight activatedaminodextran as was verified by Tyndall scatter with a focused lightbeam. These fractions were pooled to give about 10-11 ml totalsulfo-SMCC activated aminodextran in each case. A second, larger bandeluted from the column gave no Tyndall scatter. This second band wasdetermined to contain excess low molecular weight sulfo-SMCC reagent.

2. Activation of Antibody.

T3 monoclonal antibody (an anti-CD3 antibody sold by CoulterCorporation) was activated by the addition of 1.423 ml of T3 concentrate(35.14 mg/ml) to a solution comprising 0.323 ml of 2 mg/ml iminothiolanein 1X PBS and 1.587 ml 1X PBS. The resulting solution which had anantibody concentration of 15 mg/ml and an iminothiolane molarconcentration fifteen-fold larger was mixed at ambient temperature forabout one hour. The entire reaction mixture was then applied to a 100 mlG-50 Sephadex® column equilibrated with 1X PBS and the sample was elutedusing 1X PBS. First band peak fractions of about 5 ml volume werecombined to give about 10.2 ml of 4.887 mg/ml antibody solution whichcontains a total of 49.847 mg iminothiolane-derivatized T3 antibody(abbreviated as IT-T3).

3. Conjugation of sulfo-SMCC-aminodextran and

iminothiolane-T3 antibody.

5 ml of 4.887 mg/ml IT-T3 solution (about 24.435 ml antibody) were mixedwith 10.4 ml of sulfo-SMCC-1X-Amdex (about 25 mg 1X-Amdex) solution forabout two hours. In a like manner, 5 ml of 4.887 mg/ml IT-T3 solutionwere mixed with 11.2 ml of sulfo-SMCC-SX-Amdex (about 25 mg 5X-Amdex)solution. After the mixing was ended, the total volume of each mixturewas determined and 0.120 times this volume of 5 mg/ml L-cysteine in 1XPBS was added to each conjugation mixture. The L-cysteine containingmixtures were then mixed for an additional 15 minutes to effect blockingof any unreacted sulfo-SMCC moieties. Lastly, 20 mg/ml iodoacetamide in1X PBS solution in the amount of 0.120 times the total mixture volumeand 1M borate buffer solution, pH 9.8, in the amount of 0.020 times thetotal mixture volume were added to each mixture. The resulting mixtureswere mixed for about 30 minutes to block any unreacted sulfhydrylgroups.

4. Purification of anti-CD3-aminodextran conjugates.

The total volume of each conjugation mixture was reduced to about 7.5 mlby centrifuging Amicon Centripep-30 tubes containing the samples forabout 20 minutes at about 2500 rpm using a refrigerated Beckman J-6Bcentrifuge. After centrifuging, the reduced volume mixtures were placedon a Bio-Gel® A-5m agarose column (2.5 cm×48 cm) equilibrated with 1XPBS and chromatographed using 1X PBS as eluent. Eluent fractions ofabout 4 ml volume were collected using a Pharmacia LKB Frac-100collector operating in the drop collection mode. The fractions weremonitored using a LKB 2138 Uvicord S monitor operating at 280 nm. Thefirst broad band eluted from the column contained the T3antibody-aminodextran conjugate. A well-separated weaker band of lessthan half the intensity of the first band was attributed to excess IT-T3since it showed no Tyndall effect and it had the same retention time onthe column as the γ-globulin (Bio-Rad®) standard for gel filtrationcolumns. A strong, well separated third band was assigned to lowmolecular weight excess blocking reagents. Comparing conjugates madewith 1X-Amdex and 5X-Amdex, the T 3 antibody-5X-Amdex conjugate had anarrower first band which showed a longer retention time and a very weaksecond band.

The fractions collected for each conjugate were analyzed at 280 nm usinga 1 cm path length cell. Those fractions with an absorbance greater than0.160 were pooled. The yields were 55 ml of T3-1X-Amdex conjugate havinga T3 antibody concentration of 0.301 mg/ml and 54 ml of T3-5X-Amdexconjugate having a T3 antibody concentration 0.317 mg/ml. The resultsindicate that 66% and 68%, respectively, of the starting antibody wasincorporated into the conjugate products. ELISA solid phase assays forT3 in the conjugates gave a T3 antibody concentration of 0.045 mg/ml inT3-1X-Amdex and 0.112 mg/ml in T3-5X-Amdex. The lower ELISA T3 valuesrelative to the A₂₈₀ values might indicate that either that there issome blocking of active T3 antibody sites by conjugation to aminodextranor that there is some interference with the A₂₈₀ reading by an extraabsorbance contribution arising from the sulfo-SMCC reagent.

The T3-1X-Amdex and T3-5X-Amdex conjugates were also analyzed by lightscatter measurements (90°) using photon correlation spectroscopy. Thesamples were analyzed using a Coulter® N4MD sub-micron particle analyzeroperating in the molecular weight and size distribution processor (SDP)weight analysis modes. Samples of dextran T-2M, 1X-Amdex and 5X-Amdex ata concentration of 10 mg/ml were analyzed. The average molecular weightswere 3.1×10⁶ daltons (92%, dextran T-2M), 1.0×10⁶ daltons (100%1X-Amdex) and 3.5×10⁵ daltons (100% 5X-Amdex). The T3antibody-aminodextrans prepared above were concentrated to 10 ml totalvolume before light scatter measurements were made. The averagemolecular weights were 3.6×10⁶ daltons for T3-1X-Amdex and 1.4×10⁶ forT3-5X-Amdex. Using these results, the T3 antibody:1X-Amdex molar ratiowas estimated as (3,600,000-1,000,000)÷160,000=16:1 and the T3antibody:5X-Amdex molar ratio was estimated as(1,400,000-350,000)÷160,000=6.6:1.

5. Saturation T3 Antibody Conjugation to Aminodextrans.

The chromatograms of the initial preparation of T3-1X-Amdex andT3-5X-Amdex conjugates showed little, if any, excess T3 antibody whenconjugations were done using about 1:1 antibody:aminodextran weightratios. Consequently, additional experiments were performed using 3:1 T3antibody: aminodextran weight ratios and the same amount of eachaminodextran. The activation, conjugation, blocking and chromatographyprocedures were the same as those described above. However, duringchromatography, a narrow, intense band assigned to excess free IT-T3trailed the broader T3 antibody-aminodextran band in each case. TheIT-T3 band was sufficiently close to the T3-aminodextran bands so thatonly about half of the first band could be separated in each case. TheT3-1X-Amdex preparation thus yielded 75 ml of 0.576 mg/ml T3 in theT3-1X-Amdex solution and 40 ml of 0.466 mg/ml T3 in the T3-5X-Amdexsolution. ELISA solid phase assays gave 0.392 and 0.301 mg/ml T3antibody, respectively. Light scatter results indicate average molecularweights of 6.9×10⁶ daltons and 3.6×10⁶ daltons, respectively. The T3antibody:aminodextran molar ratio in the conjugates were estimated as(6,900,000-1,000,000)÷160,000= 37:1 for T3-1X-Amdex and(3,600,000-350,000)÷160,000=20:1 for T3-5X-Amdex.

III. Activation of Peripheral Blood T cells With Anti-CD3-AminodextranConjugates.

A. Isolation of Peripheral Blood Mononuclear Cells (PBMC).

The procedure used herein was performed at room temperature. Normalwhole blood was collected into tubes containing EDTA(ethylenediaminetetraacetic acid). The tubes were centrifuged at 500 gfor 10 minutes. Using sterile techniques, the buffy coat was collected,diluted 1:2 (cell:solvent) with 1X PBS and layered over Ficoll-Hypaque®.The resulting sample was then centrifuged at 400 g for 30 minutes, thePBMC interface was collected and diluted with 1X PBS. The diluted samplewas centrifuged at 300 g for 10 minutes and then washed twice with 1XPBS by resuspending the pellet, diluting with 1X PBS and centrifuging at300 g for 10 minutes. After a final resuspension in 1X PBS, the cellswere counted and cell viability was ascertained. The cells were dividedinto two parts, centrifuged as above and resuspended in the appropriatemedia for the following steps.

B. Culture and Activation of T Cells with Anti-CD3-Aminodextran.

Non-activated cells for use as the control were cultured in Nutricyte®medium containing 10% (v/v) CPSR-2 serum replacement (obtained fromSigma Chemical). The initial cell concentration was 2.5×10⁶ cells/mi.Cultures destined for T cell activation were established in the samemedium supplemented with 2-4 ng/ml phorbol 12-myristate 13-acetate (PMA)and 0.125-0.5 μg/ml of anti-CD3-aminodextran. Activated cultures wereestablished using both T3-1X-Amdex and T3-5X-Amdex. The cell suspensionswere incubated in T-150 flasks at 37° C., 5% CO₂ for 72 hours.

C. Harvesting of Control and Activated Cells.

The control cells and the T3-1X-Amdex and T3-5X-Amdex activated cellswere harvested by scraping the bottom of the tissue culture flasks witha disposable scraper, collecting the cells with a pipette and placingthem in a centrifuge tube. The cells were counted, centrifuged at 300 gfor 10 minutes and washed twice with 1X PBS as has been described.

D. Immunofluorescent Staining and Flow Cytometric Analyses of T CellActivation.

The degree of T cell activation was determined by immunofluorescentstaining of the cells, followed by flow cytometric analysis of thestained cells for the expression of both activation-associated antigens(CD71, transferrin receptor and CD25, IL2 receptor) andproliferation-associated events (DNA and proliferating cell nuclearantigen, PCNA). Aliquots containing 1×10⁶ cells of each culture wereplaced in six labelled sample tubes (3 sets of six tubes). One tube ineach culture set was stained with the following reagents which are soldby Coulter Corporation.

Tube 1: IgG2a-FITC isotype control

Tube 2: IL-2R1-FITC (IgG2a, CD25)

Tube 3: IgM-FITC isotype control

Tube 4: T9-FITC (IgM, CD71)

Tube 5: IgG1, isotype control and propidium iodide (to label DNA)

Tube 6: PCNA (IgG1) and propidium iodide.

For each set of cells (control, T3-1X-Amdex activated and T3-5X-Amdexactivated), tubes 1-4 were processed for cell surface staining byincubating the cells with the appropriate FITC conjugated monoclonalantibody for 15 minutes at room temperature. The incubated cells werethen diluted with 1X PBS, centrifuged at 300 g for 10 minutes, washedwith 1X PBS by suspension and centrifugation and finally resuspended in1 ml of 1X PBS.

The cells in tubes 5 and 6 were processed for the staining ofintracellular antigens by suspending the control cell or activated cellpellets in 1 ml of a solution containing 20 μg/ml lysophosphatidylcholine in 1% paraformaldehyde and incubating the resulting suspensionsfor 2 minutes at room temperature. After incubation, the cells wereresuspended in 1 ml of about -10° C. absolute methanol, incubated on icefor about 10 minutes and centrifuged. The resulting pellet was thenincubated in 1 ml of 0.1% NP-40 (available from Sigma Chemical) forabout 5 minutes at about 0° C., centrifuged and then incubated again forabout 15 minutes at room temperature with IgG1, (tube 5) or PCNA (tube6) monoclonal antibody. The cells were washed in 1X PBS and incubatedfor an additional 15 minutes at room temperature with goat anti-mouseimmunoglobulin conjugated to FITC. After washing in 1X PBS, 1 ml ofpropidium iodide was added to each tube.

Samples were analyzed on an EPICS® Profile I flow cytometer equippedwith a 488 nm Ar⁺ ion laser line and a power pack upgrade. Lightscattering and fluorescent signals were collected. FL1 represents theFITC fluorescent emission and FL3 represents the fluorescent emissiondue to propidium iodide. Forward light scatter (FS) was representativeof cell size and 90° light scatter (LSS=log side scatter) gives anindication of cell complexity or granularity. For single parameteranalysis (CD25 and CD71), a linear cursor was placed on the isotypecontrol histogram such that 2% of the cells were included in the cursorand the majority of cells were excluded (negative control). As a result,CD25 and CD71 positive cells were defined as those cells which emitsufficient fluorescent light to fall within the area defined by thecursor. In the dual parameter analysis for PCNA and DNA, a rectangularanalysis region was drawn on the isotype control histogram such that 2%of the cells were included within the rectangle and the majority ofcells were excluded (negative control). PCNA positive cells were,therefore, defined as those cells which emitted sufficient fluorescentlight to fall within the defined region. As the amount of DNA increases,the amount of propidium iodide taken up by a cell also increases. Thepercentage of cells in the G₀ /G₁, S, and G₂ /M phases of the cell cyclewere determined using the Multicycle® DNA analysis program.

The analytical results indicate that stimulation with T3-5X-Amdex(saturated) conjugate results in optimal T cell activation as determinedby blast cell formation, expression of cell surface activation markers(CD25 and CD71), DNA synthesis and the expression ofproliferation-associated antigen (PCNA). As can be determined by areview of FIGS. 1-4, and Table 2, the percentage of activated blastcells, as determined by increased forward and 90° light scatter, wasmaximal after stimulation with the 5X-Amdex conjugate. The results ofMulticycle® analysis of the DNA data also revealed that stimulation withthe T3-5X-Amdex conjugate resulted in the greatest percentage of cyclingcells (FIGS. 5-7 and Table 2). Similarly, the percentage of CD25, CD71and PCNA positive cells was optimal after activation with T3-5X-Amdex usshown in FIG. 8 and Table 2.

Anti-CD3 induced activation and proliferation of T cells requirescrosslinking of CD3 molecules on the T cell surface. This requirementcan be met via Fc mediated binding of anti-CD3 on the monocyte cellsurface. The data shown in Table 2 and FIGS. 1-8 was obtained frommonocyte containing cultures. T cells were purified and cultured with T3monoclonal antibody alone or with the T3 antibody conjugated to 5Xaminodextran in order to verify that the enhanced T cell activation andproliferation observed was due to the use of the 5X-Amdex conjugate.Peripheral blood mononuclear cells (PBMC) were obtained via densitygradient centrifugation over Ficoll-Hypaque® and the majority ofmonocytes were removed via adherence to plastic over a period of 90minutes. B cells and residual monocytes were depleted with B4 and Mo2conjugated magnetic beads, and the essentially pure T cells (>95% asdetermined by immunofluorescence analysis) were cultured as previouslydescribed. The results, given in Table 3, reveal that while monocytecontaining cultures are activated in the presence of T3 alone, purifiedT cell preparations are not. For both cell preparations, similaractivation was obtained with the addition of T3-5X-Amdex.

Further experiments with purified T cells verified previous findings asto the superiority of the 5X versus the 1X conjugate. The results ofsuch an experiment are given in Table 4.

                  TABLE 2                                                         ______________________________________                                        Control       CD3-1x-Amdex CD3-5x-Amdex                                       ______________________________________                                        % Blasts                                                                              7.0       43.0         60.0                                           % G.sub.0 /G.sub.1                                                                    94.7      86.4         79.4                                           % S     1.7       11.1         17.0                                           % G.sub.2 /M                                                                          3.6       2.5          3.5                                            CD25    3.0       50.0         85.0                                           CD71    2.0       33.0         73.0                                           PCNA    4.0       31.0         48.0                                           ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        % POSITIVE                                                                            T11   Mo2    B4     IL2R1 T9   % Blasts                               ______________________________________                                        PBMC                                                                          Control   84      2      10   3     4    ≦2                            +T3       83      1      15   13    4    31                                   +T3-5X-Amdex                                                                            83      2      19   14    5    40                                   Control   95      3      0    3     4    6                                    +T3       95      3      0    3     5    8                                    +T3-5X-Amdex                                                                            97      3      0    12    6    30                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        % POSITIVE                                                                                                                % S/                              T3       T11    Mo2    B4  IL2R1 T9  % Blasts                                                                             % G.sub.2 M                       ______________________________________                                        Control                                                                              78    92     2    1   3     11  5      5                               T3-1X- 6     91     3    1   42    49  41     27                              Amdex                                                                         T3-5X- 1     91     3    1   59    69  65     41                              Amdex                                                                         ______________________________________                                    

The data in the following tables correspond to the indicated figures.

                  TABLE 5                                                         ______________________________________                                        FIG. 4: Cell Cycle Data                                                       ______________________________________                                        Mean G1 = 45.7                                                                              Mean G2 = 88.1                                                                              % S = 1.7                                         CV G1 = 2.1   CV G2 = 2.1   G2/G1 = 1.928                                     % G1 = 94.7   % G2 = 3.6    Chi Sq. = 1.7                                     ______________________________________                                    

                  TABLE 6                                                         ______________________________________                                        FIG. 5: Cell Cycle Data                                                       ______________________________________                                        Mean G1 = 45.2                                                                              Mean G2 = 95.5                                                                              % S = 11.1                                        CV G1 = 10.0  CV G2 = 5.7   G2/G1 = 2.115                                     % G1 = 86.4   % G2 = 2.5    Chi Sq. = 1.6                                     ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        FIG. 6: Cell Cycle Data                                                       ______________________________________                                        Mean G1 = 46.2                                                                              Mean G2 = 96.8                                                                              % S = 17.0                                        CV G1 = 11.6  CV G2 = 6.2   G2/G1 = 2.070                                     % G1 = 79.4   % G2 = 3.5    Chi Sq. = 1.3                                     ______________________________________                                    

We claim:
 1. An antibody-aminodextran conjugate for induction of T-cellactivation and proliferation, consisting essentially of T-cell specificantibodies covalently bound to amine groups present on a water solubleaminodextran having about 7-20% by weight amine groups and a molecularweight of at least 100,000 daltons, wherein said antibodies are of asingle type each specific for the same T-cell antigen and wherein themolar ratio of said antibodies to said aminodextran is greater than orequal to two.
 2. The conjugate according to claim 1 wherein saidaminodextran has about 15-17% by weight amine groups.
 3. The conjugateaccording to claim 1 wherein said antibody is a monoclonal antibody. 4.The conjugate according to claim 3 wherein said antibody is an anti-CD3monoclonal antibody.
 5. An antibody-aminodextran conjugate for inductionof T-cell activation and proliferation, consisting essentially of T-cellspecific monoclonal antibodies and a water soluble aminodextran having amolecular weight of at least 100,000 daltons and about 7-20% by weightamine groups to which said antibodies are conjugated, wherein saidantibodies are of a single type each specific for the same T-cellantigen and wherein the molar ratio of said antibodies to saidaminodextran is greater than or equal to two.
 6. The conjugate accordingto claim 5 wherein said antibody and aminodextran are covalently linkedby a bridging group.
 7. The conjugate according to claim 5 wherein saidaminodextran contains 10-17% by weight amine groups.
 8. The conjugateaccording to claim 7 wherein said aminodextran contains 15-17% by weightamine groups.
 9. The conjugate according to claim 5 wherein saidantibody is an anti-CD3 monoclonal antibody.